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Description  |
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The subject of the invention is a system for the temperature adjustment
treatment of nucleic acids, a process for the temperature adjustment
treatment of liquid samples and a process for the identification of
nucleic acids in a sample.
The setting of a certain temperature in a liquid is an important criteria
for reactions which occur with the participation of biologically active
components. If the temperature is not correctly set, then it is possible
that a certain reaction may not take place at all or occurs to an extent
which is undesirable. This is particularly true for all reactions in which
enzymes are involved. Enzymes display temperature dependent reaction
kinetics. Furthermore, the production of complexes between biological
binding partners, e.g. complementary nucleic acids, is temperature
dependent. Nucleic acids exist in the single-stranded form above the
melting temperature and in the double-stranded form below the melting
temperature. In the event that reactions take place consecutively,
requiring different temperature regimes, it is necessary to adjust the
temperature of the reaction medium.
To date this has been achieved by transporting the reaction vessel
containing the reaction mixture back and forth between liquid baths having
different temperatures. Because of the fact that the vessel had to be
immersed in each bath for a certain period of time, the liquid to be found
therein attained the temperature of the liquid in the thermostatic media.
After a period of time appropriate for the reaction desired, the vessel
containing the liquid was transferred to another liquid bath. These
procedures were in themselves very work-intensive and difficult to
automate.
More recently devices have been developed in which the vessel containing
the liquid to be thermostatted remains confined in the one place but in
which the temperature of the thermostatting liquid is adjusted. The
disadvantage of this process is the fact that it is relatively
time-consuming because the temperature of the entire coolant has to be
adjusted. This is particularly disadvantageous in cooling processes.
Temperature adjustment treatment is employed in the nucleic acid
diagnostics field in particular. In the polymerase chain reaction
(EP-A-201184), for example, the temperature of the thermostatic medium is
varied in a cyclic fashion. For these purposes so-called thermocyclers
have been described (U.S. Pat. No. 5,038,852 and EP-A-0 488 769). In this
process a reaction block made of metal and incorporating recesses for the
reaction vessel is heated up and cooled down to effect the temperature
adjustment treatment.
In WO 92/78089 a system is described in which the liquid reactants are
contained in a closed circulatory system and are transported back and
forth between zones with cooling and heating elements. The system required
for this procedure is however complex and poorly suited for use in routine
work.
In the older, unpublished document DE-A-4409436 a process is described in
which a combined heating/cooling element is immersed in the reactant
medium and only the temperature of the reactant medium in close proximity
to the heating element is adjusted.
During the execution of a temperature adjustment treatment carried out on
liquid samples (especially during the polymerase chain reaction),
temperatures are employed at which the partial pressure of water is
relatively high. Because of this liquid usually condenses on the lid of
the reactor vessel. But because this however results in a concentration of
the reaction components in the reaction mixture which is not controllable,
it has been suggested that heating possibly be incorporated in the lid
having the purpose of revaporizing drops of liquid which have condensed on
the lid back into the gas phase. Such lid heaters are however so
positioned such that they only heat areas which do not extend into the
reaction mixture.
The objective of the invention was namely to present an alternative system
for use in the temperature adjustment treatment of liquids.
The subject of the invention is a system for the temperature adjustment
treatment of nucleic-acid-containing liquids in a vessel which has a
reusable thermostat element and a disposable heating element, whereby the
heating element is an integral part of the vessel or the vessel lid and is
dipped into the liquid during the treatment.
The invention also covers a process for the treatment of nucleic acids in a
liquid in the course of which two or more set temperatures are achieved
using a thermostat or a heating element, whereby the thermostat element is
part of a reusable device and the heating element is part of a disposable
device.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 illustrates a lid according to the invention with an integrated
heating element.
FIG. 2 illustrates the lid set onto a vessel.
FIG. 3 shows a system according to the invention in which a preparation of
nucleic acid containing liquids is conducted.
FIG. 4 shows a vessel disposed in a cooling element.
FIG. 5 shows the experimental setup of example 3.
FIGS. 6 through 12 show the results of test runs according to example 3.
The system of invention is intended for use in processes in which a liquid
or portions thereof have to be brought to different temperature levels.
This is necessary for example when processes which should take place in
the liquid, e.g. chemical or preferably enzymatic reactions occur only or
advantageously at certain temperatures. Further processes which happen to
be temperature sensitive are the above said separation of complementary
nucleic acid strands by the warming of the liquid to or the incubation of
the liquid at a temperature above the appropriate melting point (Tm) and
the creation of hybrids from the nucleic acids which are essentially
complementary to each other at temperatures which lie below the melting
point, preferably more than 15.degree. C. below the melting point, the
so-called hybridization. Another process which requires temperature
treatment at elevated temperatures is the degradation of cell
compartments. Moreover, elevated temperatures for the targeted destruction
of temperature inactivable ingredients present in the liquid, e.g. for the
inactivation of enzymes used in the degradation step (proteinases, for
example). The system of the invention enables the setting of the necessary
or desired temperature in each case regardless of how often the
temperature has to be adjusted. It is therefore also possible to
repetitively execute several or more than one of these steps consecutively
and alternately, e.g. in cycles.
In the sense of this invention, a temperature adjustment treatment of the
liquid is taken to mean one in which the liquid is so treated such that
processes which should occur in the liquid may take place at differing
temperatures. This takes into account both time-dependent temperature
profiles as well as location-dependent temperature profiles.
A prominent example for the repeated execution of treatment at differing
temperatures is the amplification of nucleic acids by means of the
polymerase chain reaction. This reaction has now been described many times
in professional circles and in various modified forms.
A prominent example of such a disclosure is U.S. Pat. No. 4,683,202. An
essential feature of the polymerase chain reaction (PCR) is the repeated
execution of cyclic temperature regimes which includes a treatment at
higher temperatures, e.g. between 90 and 95.degree. C., for reduction of
double strand nucleic acids which may be present to single strands, a
treatment at lower temperatures, e.g. between 50 and 65.degree. C., which
promotes the hybridization of primers on the nucleic acid sequences to be
amplified and a treatment at medium temperatures, e.g. 70 to 75.degree.
C., which favours the optimal elongation of the primers using the nucleic
acid to be amplified as a matrix.
The possibility of the variation of the temperature cycles are described in
EP-A-0 511 712, for example.
Nucleic acids, which may be subjected to the treatment in the sense of the
invention, are all naturally occurring nucleic bases containing
biopolymers, derivatives thereof or their analogues which can be obtained
by the modification of either the base or its sugar-phosphate backbone.
The nucleic acids may be present in the liquid in solution, in cellbound
form and may be present in a solid-surface-bound form (immobilized), e.g.
to particles.
The nucleic acids are preferably present in the solvated state at least
during the steps occurring in the course of the temperature adjustment
treatment. It is possible to bring immobilised nucleic acids into solution
and vice versa, e.g. by heating a surface-bound nucleic acid with use of
an immobilised probe.
All nucleic acid containing liquids are in principle particularly suitable
liquids, e.g. samples which are taken directly from their original
environment. Especially suitable however are liquids which have undergone
a certain amount of preparation, e.g. a step for the removal of certain
sample components (i.e. one which may interfere in the method of
analysis), the liquidisation of the sample (ie highly viscous samples), a
concentration of or dilution of the sample, a lysis step, and also the
isolation of the nucleic acids from the original sample
(pre-purification).
Liquids such as blood, urine, sputum or smears/swabs in particular are to
be taken into consideration.
The vessel in which the temperature adjustment treatment is conducted is
preferably fabricated from a material which in the course of the
temperature adjustment treatment does not release any of its components
into the liquid or deform in the course of the treatment. Particularly
suitable in this respect are plastics, e.g. polypropylene or polystyrene.
The size of the vessel is chosen such that the sample and any reagents
which may possibly be added as well as the heating element fit in.
Especially suitable are for example containers derived from Eppendorf-cups
but which however preferably do exhibit any material between the cup and
the lid. Such containers are commercially available and or may easily be
produced by injection-moulding.
A further component of the system is a lid which may be used to close the
container. It should be in the position of being able to limit the influx
and output of contaminating substances from the vessel e.g. via aerosoles,
to within acceptable levels. This too should be fabricated primarily from
temperature resistant materials as already stipulated for the containment
vessel.
A thermostat element is an object which may be actively brought to a
desired temperature and is preferably a cooling element. The cooling
element in the sense of this invention is one which is actively cooled and
can directly or indirectly transfer heat from the liquid. It does not
include the vessel. In a first embodiment of the invention the cooling
element is for example a metal block which may be cooled via Peltier
(thermoelectric) elements (dry refrigeration) or refrigerated liquids
(liquid cooling). If a metal block is employed, then this is preferably
fitted to the outer contours of the vessel. A proper fitting can be
achieved for example by the making of hollow, cylindrical recesses in the
cooling element into which the vessel may be inserted. The better the fit
of the cooling element to the external contours of the vessel, the better
is the cooling effect. In another example of the embodiment of the
invention the cooling element is preferably a metal element which projects
through an opening (which is preferably closable using the lid) into the
vessel and preferably reaches down below the surface of the liquid.
Especially cooling using a Peltier element is preferred in this respect.
In this case the cooling element is preferably protected against
contamination by the liquid by using a Teflon or polyester film. The film
is not regarded as being part of the cooling element because of the fact
that it is not reusable. The cooling element can however be in the form of
a water bath into which the vessel juts. The heat transfer from the liquid
refrigerating medium via the vessel to the reaction mixture is in this
case especially direct. A thermostat element in the sense of this
invention can certainly however have a capacity to heat, should the
temperature adjustment treatment of the liquid require that a certain
minimum threshold be respected, which lies significantly, i.e. more than
5K above room temperature. In this event it may be the case that the heat
transfer through the thermostat element to the surroundings is so large
that maintainance of the lower temperature threshold requires the addition
of heat. Nevertheless, the minimum temperature of the thermostat element
achieved in the course of the process always lies below the maximum
temperature of the heating element attained during the process.
The reusability of the element is taken to mean the possibility of using
the same cooling element to treat at least one other liquid. This other
liquid has preferably a differing composition to that of the first liquid
so that care has to be taken to minimise the contamination of the
additional liquid by the first liquid. For this reason the embodiment of
the invention in which the cooling element cools the vessel from the
outside is preferred.
A heating element in the sense of this invention is an object which is
actively heated, the development of its heat being used to warm the liquid
subject to the treatment. This can also be taken to mean a multicomponent
heater element. The heating element preferably contains a metal wire or a
metal foil, e.g. of gold, or a graphite element. Such heating elements are
known to professionals skilled in the art. The heating capacity of the
heating is designed such that the desired temperature of the liquid is
reached in the required time. This can for example be achieved by
variation of the size of the heating element or the material of
construction employed and the electrical supply.
In the sense of this invention a disposable element is taken to mean an
element which after completion of temperature adjustment treatment of a
certain liquid is disposed of (thrown away). It is not used for the
temperature adjustment treatment of any further liquids which are to be
subject to an independent temperature adjustment treatment. In the
analysis of such liquids the heating element is thrown away after each
analysis. For this reason heating elements having a simple construction
and produced at favourable cost are preferred.
An integral component of a construction element in the sense of this
invention is a component which without destruction of either the heating
element or the construction elements (vessel or lid) cannot be separated
from this element. Particularly preferred is the case when the heating
element is moulded into the vessel or the lid, this being especially
advantageous for the injection-moulding process. In the prime example the
heating can be integrated into the vessel. Care should here be taken to
ensure that the heating element is localised in the liquid receiving
region, namely, for example at the bottom of vessel or at the side walls
of the vessel which come into contact with the liquid to be heated. In the
preferred embodiment of the invention in which the heating element is an
integral component of the lid, the heating element is preferably secured
to the inside of the lid and extends into the vessel when the lid is
placed on the vessel and preferably until below the level of the liquid.
The heating element or connections, for example for electricity, extend
away on the outside from the lid and can with the use of coupling elements
be connected to a reusable device which supplies the heating element with
electricity and possibly for the regulation of the heating capacity.
The dipping of the heating elements into the vessel is in a manner that the
liquid receives an adequate amount of heat.
In addition to the system of the invention and its essential components
namely, vessel, lid, heating element and cooling element even more
suitable elements may be incorporated for the temperature adjustment
treatment of liquids and possibly succeeding further processing steps.
Construction elements are in particular for the supply of the heating and
cooling elements with electricity or coolant respectively, elements for
the adjustment of the temperature, elements for the measurement of the
temperature, transport units for the vessel, elements for the pipetting of
liquids into and out of the vessel and elements to control the whole
system. The system preferably incorporates a plurality of vessels and and
lids such that it is suitable for the treatment of several liquids in
series or parallel (particularly liquids containing nucleic acids).
There are two models possible for the heating and cooling of the liquid. In
the first model the heating element is active at intervals, for example,
when the liquid is heated over short periods (e.g. only a few fractions of
a second) while the cooling is permanently activated. Due to this,
differing and consecutive temperature gradients are preferentially set up
in the liquid, whereby the temperature in the proximity of the cooling
element remains mainly constant whilst the temperature of the liquid near
to the heating element varies to a larger extent. In so doing it is
possible to achieve a situation whereby, for example, different reactions
occur in different locations in the vessel. For example in the case that
the heating element is heated to the temperatures necessary for the
denaturation of nucleic acids (above the Tm value), denaturation of the
nucleic acid only takes place in the proximity of the heating element.
Thereafter the denatured nucleic acids can be transported to an area in
which hybridization with other nucleic acids can take place. The transport
can occur by way of a convection mechanism but diffusion is favoured. In a
second particularly preferred embodiment of the invention, the cooling and
heating functions are continually activated and preferably remain
constant. Over a sufficiently long period of time in this case a stable
temperature gradient is achieved which because of the heat conducting
capacity of the liquid as well by diffusion and possibly convection is
controlled in the liquid. Also in this case different reactions may occur
in different locations in the vessel. In this model all components which
are to take part in the respective reaction are preferably in solution.
In one preferred model the system consists of a plurality of lids, a
plurality of vessels and essentially a thermostatted block as well as
elements active in the supply and control of electrical energy for the
operation of the heating element.
The thermostat block is preferably a metallic body having receival
bore-holes for plastic containers. The thermostatic effect necessary is
provided by use of a thermostatted liquid (heat-transfer liquids,
circulation refrigeration), employment of Peltier elements or other known
thermostatting processes.
The dimensions of the bores for the plastic containers are fitted exactly
to the external contours of the plastic vessel because direct contact with
the thermostat block is necessary for the effective transfer of heat
required.
Such construction features are however known to professionals skilled in
the art.
The depth of the bore-holes should preferably be in the relation of 5:1 to
the diameter because this ensures that when a temperature gradient is well
set up, a mixing of the liquid takes place which is favourable for the
system.
The plastic vessel in which the temperature adjustment treatment
continually takes place is preferably made of polypropylene and has a wall
thickness of less than 1.0 mm (but which depends on the total volume of
the reaction mixture).
In the execution of reactions typical in Clinical Chemistry and Nucleic
Acid Diagnostics, volumes of less than 1 ml are normally employed. This
dictates that the approximate dimensions of the vessel with the lid (which
is also manufactured using the injection-moulding process) are 8 mm (inner
diameter) and 40 mm in height, respectively.
The disposable heating element consists preferably on the whole of a
plastic moulded form, the electrical connections and the heat transfer
film. The dimensions of the disposable heating element are fitted to the
dimensions of the reaction vessel.
A preferred embodiment of the disposable heating element is one in which a
prefabricated arrangement of contacts and heat transfer film is integrated
into a plastic component produced by injection-moulding consisting of a
lid and a mount.
The heat transfer film is preferably a 20 .mu.m thick gold film. The
injection-moulded plastic component is made of polypropylene. The area of
the heating element is preferably 60 mm.sup.2 and the lower end of the
element extends to the bottom of the vessel in the reaction vessel.
The system detailed above for the temperature adjustment treatment of
nucleic acid containing liquids can be employed to advantage in many ways.
Also an object of this invention is therefore a process for the treatment
of nucleic acids in a liquid with the application of two or more
temperatures using a cooling or heating element whereby the cooling
element is an integral part of a reusable device and the heating element
is part of a disposable arrangement. The above-mentioned features are also
valid for this process. The application of the process of the invention to
thermocyclic reactions has proven to be particularly practical. In the
course of such processes different reactions take place at different
temperatures. The reactions can take place by subjecting the reagents to
certain temperatures. This can on the one hand, as described above, be
achieved by time-dependent variation of the temperature profile in the
reaction mixture and by increasing or decreasing the heating or cooling
capacity respectively or, on the other hand, however also by stipulating a
constant temperature profile between the heated and cooled regions.
Integrades are also conceivable eg achieved by convection in the mixture.
Of importance is that, in accordance with the process of invention, the
reactants of the desired reaction are consecutively subjected to different
temperatures so that the desired reactions can take place. During the
entire period of treatment a cooling effect can be achieved by control of
the heating and cooling capacities for example in cyclic reactions so that
the reactants are subjected to different reaction parameters in a cyclic
fashion and therefore reaction cycles can be conducted consecutively. By
subjecting the reaction mixture to a relatively constant temperature
gradient, a cyclic treatment occurs favoured by diffusion of the reaction
partners from a first spacial volume segment of the reaction mixture
having one temperature to a second spacial volume segment having a second
temperature. The cyclic course of events occurs by diffusion in a spacial
volume segment with one temperature as required by the succeeding reaction
(e.g. renewed by the first or a third temperature). Because diffusional
processes normally occur relatively slowly, it is preferable to select a
rather steep temperature gradient thereby ensuring that the temperature
drops between the heating element and the cooling element are limited to a
comparatively short path. Typical pathlengths between the heating element
and the cooling element are of a few millimeters.
A typical example of a process for the temperature adjustment treatment of
nucleic acids is the amplification of nucleic acids or parts thereof. One
example of this is the polymerase chain reaction as described in U.S. Pat.
No. 4,683,202. One further example is the ligase chain reaction.
A further object of the invention is a process for the detection of nucleic
acid in a sample by
a) Liberation of the nucleic acid which is to be detected from compartments
in which it is contained in a vessel
b) Replication of sequence information which is derived from the presence
of nucleic acid in the vessel
c) Determination of the sequence information whereby the nucleic acid is
not removed from the vessel during and between the steps a) to b).
The system of the invention can in so doing be used to significantly
simplify the nucleic acid determination procedure. Particularly preferred
is the case when the liquid is not transported within the vessel from one
location to the other (excepting mixing procedures).
The liberation of the nucleic acids can take place in principle by means
which are known. Usual treatments consist of the lysis of cell walls, e.g.
with suitable reagents such as proteinase K, detergents or alkali or/and
heat. This results in the salvation of the nucleic acids and makes them
accessable for reagents which further process them. This step takes place
in a vessel which is inert under the conditions of the reaction and the
succeeding steps b), e.g. polypropylene. In the same vessel sequence
information which is derived from the presence of nucleic acid is
replicated e.g. by amplification of a segment of the nucleic acid
liberated. This can occur using the polymerase chain reaction.
The term sequence information is taken to mean a sequence of bases eg one
(nucleotide sequence) which is part of or the entirety of nucleic acid to
be determined.
In principle though the sequence information can be contained in a
nucleotide which has been coupled by cross-linking to the nucleic acid to
be determined and finally replicated. This can for example be to do with a
so-called signal amplification. For the object of the invention it is
essential that the reactions taking place in step a) and b) occur in the
same vessel. Step b) can for example be initiated when the nucleic acid
containing liquid in the vessel is subjected to a temperature adjustment
treatment with the aid of the above-mentioned reusable thermostat element,
in particular the cooling element, and the disposable heating element.
This can preferably occur when the vessel during steps a) and b) is stored
in the reusable cooling element and for the execution of step b), the
disposable heating element is introduced into the vessel. If the
disposable heating element is integrated into the lid, this can be already
situated on the vessel during step a) and be engaged in the heat treatment
and also be engaged after release of the nucleic acid for the purposes of
heat treating.
The determination of the sequence information can in principle occur by the
use of procedures known to professionals skilled in the art e.g. by
transferring the reaction mixture from step b) into a container in which
the nucleic acids produced preferably in the course of a hybridisation
reaction can be determined. One possible experimental procedure employs
the so-called sandwich principle as described in EP-B-0 079 139. This
procedure uses a capture probe complementary to a first part of the
replicated sequence information which is either or can be bound to a
solid-phase support and a detector probe which is labelled and is
complementary to another part of the replicated sequence information. The
production of the complex of probe and replicated sequence information
containing nucleic acid is interpretated as being an indication of the
presence of nucleic acids in the sample. The avoidance of the transfer of
nucleic acid from one vessel to the other markedly reduces the risk of
contamination of the reaction mixture and the surroundings. Furthermore,
the process is much simpler and can be conducted using much less
equipment.
FIG. 1 illustrates a lid (1) according to the invention with an integrated
heating element. It is perceivable that the lid has a closable part which
is fitted exactly to the shape of the opening of the vessel which is to be
sealed. The seal extends to a plastic mounting (6) for the heating element
(5). The heating element is secured to the surface of this plastic mount
in such a manner that the power supply wiring (4) for the heating element
can rest inside the plastic mount or seal and at one end electrical
contacts (2) extend far enough to a power supply.
The lid is shown in FIG. 2 such that it is actually set onto a vessel. The
outer dimensions for a vessel are displayed in FIG. 2 and are those for
the lid shown in FIG. 1. These external dimensions are suitable for the
execution of the process of invention eg for the amplification process but
can however be easily adjusted to differing amounts of liquid in
particular by a professional skilled in the art. The number 7 in the
figure denotes the vessel.
In FIG. 3 a system according to the invention with a sample preparation
module 17 is shown, the said being one in which a preparation of nucleic
acid containing liquids for amplification can be carried out and in which
the amplification itself can be conducted. In this figure the top handling
arm (lid handling arm 11) can grip the lid shown in FIG. 1 (top 1) and put
this onto reaction vessels (disposable devices 12). Furthermore, contacts
for the electricity supply to the lid heater are integrated into the top
handling arm. With the aid of the pipetting unit and pipette tips
(disposable tips 13), reagents 14 and/or sample liquid 15 can be
transferred to the reaction vessel 7 (disposable device 12 in this case).
As soon as the lid of the invention is spent, it can be transferred to the
waste bin 16 (solid phase disposable with top). All steps involved are
preferably conducted in equipment which allows movement in all 3
dimensions (x,y,z) and in which pipetting stages and transport steps can
be executed (e.g. laboratory robot 18).
FIG. 4 illustrates a system with a power supply (8), electrical contacts
(2), a vessel (7) contained within a cooling element (10), and a reaction
mixture (9) which is mixed by the development of heat by the heating
element in a convective manner, as indicated by the arrows.
FIG. 5 shows schematically a system for conducting the experiment including
a temperature adjustment treatment. Control unit (19) controls the heating
element (5), while computer (20) is used for the control of and
calculations for the entire process.
Reference Numerals
1. Lid with disposable heating element
2. Electrical contacts
3. Lid seal
4. Electrical supply wiring for the heating element (moulded inside the
plastic)
5. Heating element (gold foil)
6. Plastic mount for the heating element
7. Vessel
8. Connection for power supply
9. Reaction mixture
10. Cooling element
11. Lid handling unit (picking-up, taking-off, putting-on of lid,
electricity supply via contacts)
12. Disposable device (contains a plurality eg 16 vessels, which are linked
to each other)
13. Pipette tips on a pipetting arm of the device
14. Reagents in vessel
15. Sample liquid in vessel
16. Waste bin for lid
17. Sample preparation module (receiver for vessels, thermostat block)
18. Laboratory robot (control of transport and thermostatting steps and
operating procedure)
19. Control unit for the heating element
20. Computer for the control of and calculations for the whole process
EXAMPLE 1
Establishment of a system to conduct a DNA analysis
The system is comprised of a waterbath which is thermostatted at 57.degree.
C.
Above the waterbath is an apertured plate secured at a certain distance and
which enables the reaction vessel as shown in FIG. 2 to extend into the
water to an extent of about half its length. A rim on the vessel prevents
the slipping of the vessel into the water. The aperture plate receival
bores are only marginly larger than 8 mm. The plastic vessel is made of
propylene having a wall thickness of 0.4 mm (FIG. 2).
The inserted heating element is shown schematically in FIG. 1. It is an
injection-moulded plastic component which incorporates a 20 .mu.m thick
gold foil and wiring integrated in such a manner that the liquid can wet
the gold foil on one side.
EXAMPLE 2
Execution of a DNA-analysis under conditions of static temperature gradient
1. Sample preparation/DNA Isolation
Human leucocytes were isolated from whole human blood using the following
method and employing the QIAamp Blood Kit (Cat. No. 29104), Quiagen (FRG,
P.O. Box., 40719 Hilden).
200 .mu.l EDTA-anti coagulated whole blood, 25 .mu.l proteinase-K solution
(19 mg/ml) and 200 .mu.l degraded sample were pipetted into a 2 ml
Eppendorf container. The sample was immediately shaken with Vortex.RTM. to
resuspend the pellet which was forming in solution. The sample was heated
for 10 minutes at 70.degree. C., cooled to room temperature. 210 .mu.l
isopropanol were then added to the solution. The sample was transferred to
a QIAamp "spin-column". The spin-column is a centrifuging device/tube
which is open downwards and to which a glass fiber fleece is attached at
the bottom.
The spin-column was inserted into a sample collection container (2 ml
Eppendorf container) and centrifuged in a benchtop centrifuge at
6000.times.g for 1 minute. The filtrate was discarded and 500 .mu.l
washing buffer was pipetted into the spin column. It was then centrifuged
1 minute at 6000.times.g. The filtrate was discarded and the washing
procedure was repeated.
Thereafter 200 .mu.l elution solution (10 mM Tris/HCl, 1 mM EDTA, pH 8. was
pipetted into the spin column and the bound DNA was eluted out of the
glass fiber felt after renewed centrifugation (1 minute, 6000.times.g).
The purified DNA was characterised using gel electrophoresis and photometry
(absorbance maxima at 260 nm and 280 nm). Typically 6 .mu.g DNA in 200
.mu.l elution solution (approx. 30 ng DNA/.mu.l) having an extinction
coefficient A60/270 from 1.7-1.9 (an extinction of 1000 mE at 260 nm
corresponds to a sample DNA content of 50 ng/.mu.l) were obtained from 200
.mu.l whole blood (approx. 5.times.10 leucocytes per ml).
The fragment size of the DNA eluted was between 1 and 50 Kbp and mainly
between 20 and 40 Kbp as determined using gel electrophoresis (1% agarose
gel, ethidium bromide staining).
2. Amplification/DNA replication
A sequence from the human-tPA-gene (tPA=tissue-type plasminogen activator)
was amplified using two specific primers. The sequences of the primers
employed were:
Forward (i.e. upstream) 5'-AGA CAG TAC AGC CAG CCT CA-3'SEQ ID No: 1!
Reverse (ie downstream) 5'-GAC TTC AAA TTT CTG CTC CTC-3'SEQ ID No: 2!
A 375 bp length amplified segment results using these primer pairs.
The following mastermix was pipetted into the polypropylene (PCR)-reaction
vessel described above:
10 .mu.l 10 fold PCR-buffer with MgCl.sub.2 (100 mM Tris/HCl pH 8.9; 500 mM
KCl, 15 mM Mg Cl.sub.2)
2 .mu.l 10 mM dNTP-mix (ie 10 mM dATP, dGTP, dCTP and dTTP each)
0.5 .mu.l Taq-polymerase (5 U/.mu.l)
1 .mu.l Forward primer (30 .mu.M, for sequence see above)
1 .mu.l Reverse primer (30 .mu.M, for sequence see above)
82.5 .mu.l autoclaved, double-distilled water
All the reagents used in the amplification (except for the primer) are out
of the PCR Core Kit (Cat. No. 1578 553) from Boehringer Mannheim.
The mastermix was placed briefly on a Vortex.RTM. stirrer, then centrifuged
in a benchtop centrifuge. 3 .mu.l of sample containing DNA (from point 1,
DNA content approx. 30 ng/.mu.l) were pipetted to the mastermix. The PCR
vessel was placed in a heating/cooling block of the invention and sealed
with a disposable heating element containing lid.
The heating element was arranged in such a manner that the resistance wire
extended about two thirds of the way into the PCR mix. The heating element
was connected to the electricity supply and the PCR mix incubated for 0.5
hours such that the resistance wire was kept at a temperature of
95.degree. C. and the tube inner wall attained a temperature of 58.degree.
C.
After completion of the amplification, the PCR-mix was analysed according
to point 3.
3. Analysis of the amplified DNA
10 .mu.l of the amplified PCR sample were applied to the application site
of a 1% agarose gel described in point 2. 800 ng of Boehringer DNA Langen
standard VI (Cat. No. 1062 590, fragment size 2176 bp to 154 bp) were
applied.
The gel was developed in an electric field for 2 hours and then analysed on
a UV table.
In the presence of human leucocyte DNA in the mastermix, an intense DNA
band was visible in the gel (375 bp) and was located between the 394 bp
band and the 298 p-band of the Langenstandard VI.
EXAMPLE 3
Temperature adjustment treatment with time-variable temperature gradients
The aim of the experiment was determination and optimalisation of a
periodically varying temperature gradient using the system of the
invention. For these purposes a test tube made of polypropylene and filled
with 300 .mu.l autoclaved, double distilled water was used and inserted in
the metal thermostat block which was cooled with a Peltier element. A
commercially available Pt24-Chip was integrated into the lid which
simultaneously served the heating element and the temperature probe. The
heating element extended into the water. In a neighbouring test tube the
temperature setting of the thermostat block was monitored using a M
4011BBC temperature sensing device. The experimental set up is depicted in
FIG. 5. The unit was operated using varying temperature intervals. The in
operation time is defined as the time interval in which the heating is
active and the out of operation time was defined as the time interval
between the heating cycles. The results of the tests are given in FIGS. 6
to 12. It is evident that the test run according to FIG. 12 does not
facilitate a sensible temperature adjustment treatment because the time
intervals are probably long enough for rehybridization of the nucleic
acids. On the basis of these tests a professional skilled in the art can
determine the best conditions for his own special system (special
geometry, heating rate etc.).
TABLE 1
______________________________________
Stand Temp./.degree. C.
Measured Temp./.degree. C.
On/ms Off/ms
FIG.
______________________________________
4 4.9 800 2000 FIG. 6
10 11.0 400 2000 FIG. 7
10 10.6 800 2000 FIG. 8
10 11.0 800 3000 FIG. 9
10 10.9 800 4000 FIG. 10
20 20.2 800 3000 FIG. 11
20 20.5 2000 1000 FIG. 12
______________________________________
__________________________________________________________________________
# SEQUENCE LISTING
- (1) GENERAL INFORMATION:
- (iii) NUMBER OF SEQUENCES: 2
- (2) INFORMATION FOR SEQ ID NO: 1:
- (i) SEQUENCE CHARACTERISTICS:
#pairs (A) LENGTH: 20 base
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
- (ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc - # = "Oligodesoxyribonucleotide"
- (iii) HYPOTHETICAL: NO
#1: (xi) SEQUENCE DESCRIPTION: SEQ ID NO:
# 20 CTCA
- (2) INFORMATION FOR SEQ ID NO: 2:
- (i) SEQUENCE CHARACTERISTICS:
#pairs (A) LENGTH: 21 base
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
- (ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc - # = "Oligodesoxyribonucleotide"
- (iii) HYPOTHETICAL: NO
#2: (xi) SEQUENCE DESCRIPTION: SEQ ID NO:
# 21 TCCT C
__________________________________________________________________________
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